PROJECT SUMMARY/ABSTRACT Understanding the mechanisms which drive immune responses to autoantigens is of high priority. In rheumatoid arthritis (RA), protein citrullination is a major target of the immune system. Significant data implicates the anti-citrullinated protein immune response in RA pathogenesis, with recent evidence suggesting that mechanisms which enhance citrullination are associated with the worst disease outcome. While different mechanisms likely promote citrullination in RA, our published and preliminary data suggest that citrullination and the complement system are interacting pathways that amplify each other to enhance autoantigen production and immune-mediated damage in a sizable subset of RA patients. Thus, we have found that cellular membranolysis mediated by complement is a potent inducer of protein citrullination in the RA joint, and that this process is associated with citrullination of critical regulatory components of the complement system (complement factor H (CFH), and C3), enhancing complement activation. The previous finding that anti-CFH antibodies are prevalent in RA further highlights the importance of CFH in this disease, adding an extra mechanism that may dysregulate complement in RA. Taken together, we propose that citrullination-induced complement dysregulation and anti-CFH antibodies are key amplifiers that propagate damage and citrullination activity in the RA joint. We will examine this hypothesis in 3 Specific Aims. In Aim 1, we will use mass spectrometry and biochemical assays to define the structural and functional effects of citrullination on CFH and C3, as well as the effect of these modified proteins on the induction of the terminal complement complex, neutrophil hypercitrullination and B cell activation mediated by citrullinated C3dg. Aim 2 will use multiple reaction monitoring (MRM) to quantify citrullinated CFH and C3 in synovial fluid from RA and other arthritides to define their association with complement deposition and lytic activity in the joint. Aim 3 will determine whether anti-CFH antibodies participate in the dysregulation of complement in RA by defining their prevalence and clinical significance in RA, their association with complement activation, and their effect on CFH function. These studies will define novel interacting mechanisms of disease amplification relevant to sustain inflammation and aggravate damage in the rheumatoid joint. Furthermore, the studies will show that these pathways are active in vivo in RA, identifying markers and subgroups for precise therapeutic intervention.